Plk3, A New Player In The Hypoxia Regulatory Networ.

描述（由申请人提供）：HIF-1控制许多涉及癌症生物学关键方面的基因的转录。HIF-11是HIF-1对缺氧反应的诱导亚基，其过表达是许多癌症的预后因素。PI3K、PDK1、Akt介导的主要信号轴（也称为蛋白激酶B）在HIF-11表达调控中起重要作用。已知GSK32和MAPKs都能直接磷酸化HIF-11，从而影响其稳定性和/或核定位。为了了解抑制肿瘤发展和肿瘤血管生成的调控网络，我们最近发现HIF-11是polo样激酶3 （Plk3）的一个新的体外底物。Plk3在体外强烈磷酸化HIF-11的丝氨酸576和657，这两个残基分别位于氧依赖降解区域和靠近核输出信号。通过对原代等基因小鼠胚胎成纤维细胞（mef）的研究，我们发现PLK3-/- mef在缺氧或模拟缺氧的镍处理下对HIF-11的诱导高度敏感。与野生型mef相比，PLK3-/- mef含有高水平的Akt1/PKB活性，这与GSK32的抑制性磷酸化密切相关。与Plk3在调节缺氧信号网络中的潜在作用一致，Plk3 -/-小鼠在高龄时在各器官发生肿瘤，Plk3 -/-肿瘤体积大，血管化程度高，表明肿瘤血管生成活跃。基于这些关于Plk3和HIF-11之间物理和功能相互作用的观察，我们假设Plk3负调控缺氧调节网络和HIF-11依赖性肿瘤血管生成。为了验证这一假设的有效性，我们将(i)研究Plk3与已知信号分子（包括磷酸化HIF-11的GSK32和MAPKs）之间的功能相互作用，并确定Akt1上游的其他Plk3靶点，（ii）确定Plk3 -/-小鼠在缺氧条件下是否容易发生肿瘤，（iii）研究携带HIF-11的Plk3磷酸化抗性等位基因突变的小鼠在镍暴露后是否更容易发生肿瘤。体外和体内联合研究将极大地促进阐明Plk3在缺氧反应或暴露于环境致癌物（如镍化合物）后调控HIF-11的新机制。详细了解HIF-11的分子调控将显著增加现有的肿瘤血管生成和肿瘤细胞对抗癌治疗的耐药性知识。

DESCRIPTION (provided by applicant): HIF-1 controls the transcription of many genes that are involved in key aspects of cancer biology. Overexpression of HIF-11, an inducible subunit of HIF-1 in response to hypoxia, is a prognostic factor in many cancers. The major signaling axis mediated by PI3K, PDK1, and Akt (also referred as protein kinase B) plays a significant role in the regulation of HIF-11 expression. Both GSK32 and MAPKs are also known to directly phosphorylate HIF-11, thereby affecting its stability and/or nuclear localization. To understand the regulatory network that suppresses tumor development and tumor angiogenesis, we have recently identified HIF-11 as a new in vitro substrate of Polo-like kinase 3 (Plk3). Plk3 strongly phosphorylates HIF-11 on serines 576 and 657 in vitro, two residues that lie in the oxygen-dependent degradation domain and near the nuclear export signal, respectively. By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3-/- MEFs are hyper-sensitive to the induction of HIF-11 under hypoxia or treated with nickel, a hypoxia mimetic. Compared with that of wild- type MEFs, PLK3-/- MEFs contain a high level of Akt1/PKB activities, which is tightly associated with the inhibitory phosphorylation of GSK32. Consistent with the potential role of Plk3 in regulating the hypoxia signaling network, PLK3-/- mice develop tumors in various organs at an advanced age and PLK3-/- tumors are large in size and highly vascularized, suggesting active tumor angiogenesis. On the basis of these observations regarding physical and functional interactions between Plk3 and HIF-11, we hypothesize that Plk3 negatively regulates the hypoxia regulatory network and HIF-11-dependent tumor angiogenesis. To test the validity of this hypothesis, we will (i) study functional interaction between Plk3 and known signaling molecules, including GSK32 and MAPKs that phosphorylate HIF-11, and identify additional Plk3 target(s) upstream of Akt1, (ii) determine whether PLK3-/- mice are prone to tumorigenesis under hypoxia, and (iii) investigate whether mice harboring Plk3 phosphorylation-resistant mutant alleles of HIF-11 are more susceptible to tumorigenesis after nickel exposure. The combined in vitro and in vivo studies will greatly facilitate the elucidation of a new mechanism by which HIF-11 is regulated by Plk3 during hypoxic responses or after exposure to environmental carcinogens such as nickel compounds. A detailed understanding of the molecular regulation of HIF-11 will add significantly to the existing knowledge of tumor angiogenesis and tumor cell resistance to anti-cancer therapies. PUBLIC HEALTH RELEVANCE: Polo-like kinases function to regulate various aspects of cell growth and cell death. Our recent biochemical and mouse genetic studies show that Plk3 plays an important role in the regulation of the HIF- 11 signaling pathway, as well as in suppressing tumorigenesis. The major goal of this project is to elucidate what signaling pathways mediate Plk3 function in hypoxic stress responses and how genotoxic metal toxicants usurp cellular mechanisms that regulate HIF-11 during hypoxia.